842 THE COMPLEMENT FIXATION TEST FOR SYPHILIS 



persons free from syphilis is added. This added serum must contain complement and natural 

 antisheep amboceptor which is determined by titration. 



The antigen, prepared according to the method of Bordet and Ruelens' from calf heart 

 extracted first with acetone and then with alcohol, is used in o.i-cc. and 0.2-cc. amounts. The 

 alcohol is not evaporated prior to use. 



In accordance with the procedure recommended by Weinberg, one hour at 37° C. is al- 

 lowed for fixation. 



A 5 per cent suspension of sheep blood corpuscles, washed once or twice and restored to 

 the original volume, is used in the hemolytic system. The amount used varies with each 

 serum, being one-third of the greatest volume which is hemolyzed by o.i cc. of the serum. 



METHOD USED BY RENAUX IN THE PASTEUR INSTITUTE, BRUSSELS^ 



The patient's serum is inactivated for twenty minutes at 56° C. and is used in amounts of 

 0.1 and 0.05 cc. 



One drop, approximating 0.04 cc, of fresh guinea pig serum is used as complement. This 

 is titrated alone and in 75 per cent and 50 per cent dilutions. These dilutions are tested with 

 cells sensitized with varying amounts of amboceptor. After twenty minutes at 37° C, the 

 most satisfactory complement dilution is chosen. 



One antigen, that described by Bordet and Ruelens, is used. It is an alcoholic extract 

 of the acetone-insoluble lipoids of calf heart. To dilute it, i cc. of extract is evaporated to 

 dryness at 37° C, after which the residue is suspended in 3 cc. of distilled water and then 

 27 cc. of 0.9 per cent salt solution are added. Four drops or 0.16 cc. are used in the test. 



One hour at 37° C. is allowed for fixation. 



Antigoat rabbit serum inactivated for thirty minutes at 56° C. is employed as ambo- 

 ceptor. The preliminary titrations are made with blood cell suspensions sensitized with differ- 

 ent strengths of the amboceptor varying from i : 20 to i : 60 dilutions. These dilutions are 

 tested with both diluted and undiluted complement. The most satisfactory dilution of com- 

 plement is chosen after twenty minutes at 37° C, and then a second amboceptor titration is 

 performed. This consists of four drops of antigen and one drop of complement with one, 

 two, and three drops of the cells sensitized as previously described. After thirty minutes, the 

 degree of sensitization is chosen which gives complete hemolysis with one drop of corpuscles, 

 almost complete with two drops, and marked hemolysis with three drops. One drop of 

 sensitized cells is used in the test. 



Defibrinated goat blood, washed three times, restored to its original volume, and then 

 diluted to twice this volume with 0.9 per cent salt solution, is employed. 



METHOD USED BY NOGUCHI, ROCKEFELLER INSTITUTE, NEW YORK CITY^ 



Either 0.02 cc. of fresh serum or 0.04 cc. of serum inactivated for about fifteen minutes 

 at from 55° to 56° C. is used. 



The complement consists of guinea pig serum titrated in the presence of fixed amounts of 

 cells and amboceptor. Two units, usually o.i cc. of a 40 per cent dilution, are used. 



As an antigen, an alcoholic extract of acetone-insoluble lipoids is used. Antigens arc 



'Bordet, J., and Ruelens, G.: "L'Antigene syphilitique de I'lnstitut Pasteur de Bruxelles," 

 Compt. rend. Soc. de biol., 82, 880. 1919. 



^ Investigations on the Serodiagnosis of Syphilis, League of Nations, Health Organization, C. 5 

 M. 5 III. 1924. 



3 Noguchi, H.: Serum Diagnosis of Syphilis and Luetin Reaction (3d ed.). Philadelphia: J. B. 

 Lippincott Co., 191 2. Dr. Noguchi furnished antigen only for the standardization tests made in 

 1925. See Gilbert, R., and Langworthy, V.: "Standardization of the Wassermann Test," Am. J. 

 Syph., 10, loi. 1926. 



